Chromatography separates the individual components contained within a sample so that they may be identified. For example, in liquid chromatography two phases are involved, a mobile phase and a stationary phase. A liquid sample mixture (the “mobile phase”) is passed through a column packed with particles (the “solid phase”) in order to effect a separation of the constituent components. The particles in the column may or may not be coated with a liquid designed to react with the mobile phase. The constituent components in the mobile phase, i.e. in the sample, pass through the packed column at different rates based upon a number of factors. The separation of the sample into its constituent components is then analyzed by observing the sample as it exits the far end of the column.
The speed with which the different constituent components pass through the column depends on the interaction of the mobile phase with the solid phase. The components in the sample may physically interact with the particles or a substance coating the particles such that their movement through the column is retarded. Different components in the sample being analyzed will react differently to the particular particle and/or coating by interacting with the particular particles and/or coating with differing degrees of strength depending upon the chemical makeup of the component. Those components which tend to bond more strongly to the particles and/or coating will pass through the column more slowly than those components which bond weakly or not at all with the particle/coating. In addition to chemical reactions, the size of the components in the sample may dictate the speed with which they pass through the column. For example, in gel-permeation chromatography, different molecules in the solution being analyzed pass through a matrix containing pores at different speeds thereby effecting a separation of the different molecules in the sample. In size exclusion chromatography the size of the particles and their packing method in the column combine with the size of the components in the sample to determine the rate at which a sample passes through the column (as only certain size components may easily traverse the gaps/interstitial spaces between particles).
The separated sample travels into a detector at the far end of the column where the retention time is calculated for the various components in the sample. The retention time is the time required for the sample to travel from the injection port (where the sample is introduced into the column) through the column and to the detector. The amount of the component exiting the solid phase may be graphed against the retention time to form a chart with peaks which are known as chromatographic peaks. The peaks identify the different components.
The separated components may be fed into a mass spectrometer for further analysis in order to determine their chemical make-up. Systems with two mass spectrometer stages are referred to as LC-MS-MS systems. A mass spectrometer takes a sample as input and ionizes the sample to create positive ions. A number of different ionization methods may be used including the use of an electronic beam. The positive ions are then separated by mass in a first stage separation commonly referred to as MS1. The mass separation may be accomplished by a number of means including the use of magnets which divert the positive ions to differing degrees based upon the weight of the ions. The separated ions then travel into a collision cell where they come in contact with a collision gas or other substance which interacts with the ions. The reacted ions then undergo a second stage of mass separation commonly referred to as MS2.
The separated ions are analyzed at the end of the mass spectrometry stage or stages. The analysis graphs the intensity of the signal of the ions versus the mass of the ion in a graph referred to as a mass spectrum. The analysis of the mass spectrum gives both the masses of the ions reaching the detector and the relative abundances. The abundances are obtained from the intensity of the signal. The combination of liquid chromatography with mass spectrometry may be used to identify chemical substances such as, for example, metabolites. When a molecule loses electrons, covalent bonds often break, resulting in an array of positively charged fragments. The mass spectrometer measures the masses of the fragments which may then be analyzed to determine the structure and/or composition of the original molecule. The information may be used to isolate a particular substance in a sample.
Metabolism may be defined as the chemical changes that take place in a cell or an organism that are used to produce energy and the basic materials which are needed for life processes such as mitosis. The byproducts of the chemical reaction may be referred to as metabolites. By analyzing and identifying the metabolites that are present in a sample, it is possible to determine the route of metabolism. For example, an analysis of metabolites in urine may be used to determine what substances were ingested by the individual that produced the urine. The identification and analysis of the metabolites is often performed using liquid chromatography in combination with mass spectrometry.
Conventionally, the analysis of metabolites involves three separate sample runs. The first sample run is a control. Following the control sample run a first analyte sample run is conducted. The chromatographic peaks from the analyte sample results are compared to the chromatographic peaks of the control and the results of the comparison are used to eliminate the components that appear in both samples. A second analyte sample run is then conducted that focuses on the components unique to the analyte sample in order to identify unexpected metabolites that appear in the analyte sample but not in the control sample. Unfortunately, the comparison of the control sample to the first analyte sample is a time intensive procedure requiring in most cases direct human participation.